Thief of Time

A bee with a miniaturised transponder attached to its thorax to enable monitoring of its flight after anaesthesia.

By Guy Warman, Craig Millar & James Cheeseman

General anaesthesia alters our perception of time by shifting the expression of clock genes to a new time zone, leading to chemically induced jet lag.

Despite the astonishing fact that 234 million general anaesthetics are administered each year around the world, the way in which anaesthetics put you to sleep remains unknown. The phrase “put you to sleep” is synonymous with general anaesthesia, and recent evidence suggests this metaphor may be more accurate than we had ever imagined. At least some anaesthetics appear to act in part by hijacking sleep-promoting pathways in the brain to exert their effects.

While there are obvious similarities between natural sleep and general anaesthesia, there are also marked differences. Sleep is an active process in which our brain changes between states of high activity (REM sleep) and low activity (slow wave sleep). During general anaesthesia, the brain’s activity resembles only slow wave (or deep) sleep.

Another obvious but important difference is that while pain will waken someone from sleep it does not rouse the anaesthetised patient.

The intriguing phenomenon we have sought to explain is the common feeling after anaesthesia that time has not passed. While we often wake up just before our alarm sounds in the morning, aware that time has passed during the night, when people emerge from general anaesthesia they often report they have only just been put to sleep.

Our current lack of understanding of the effects of general anaesthesia on time perception is attributable in part to the difficulty of investigating this phenomenon in humans, who are notoriously bad at accurately determining the time of day without the assistance of a watch or clock.

The honey bee provides a unique and powerful model with which to study the effects of anaesthesia on the perception of time. Bees exhibit an extraordinary array of complex and accurately timed behaviours in their normal daily life. They have an ability to continuously consult their circadian clock as we might a wrist watch to determine the time of day. This enables a bee to time its foraging visits to flowers so that they coincide with periods of maximum nectar production; to navigate using the position of the sun in the sky (known as ‘sun-compass orientation’); and to inform her siblings in the hive of the correct position of a food source via the famous waggle dance (Fig. 1).

Figure 1. Bees use their sense of time to navigate to flowers at the time of the day when nectar is produced. (a) A bee visits a flower east of the hive in the morning. At 8am the bee will fly towards the sun, but over the course of the day as the sun appears to move in the sky, her angle of orientation with respect to the sun will change. If the nectar source is particularly profitable she will perform a waggle dance inside the hive (b) to inform her siblings of the correct angle of the food source with respect to the sun. This dance can continue for several hours, and once again the bee’s sense of time is sufficiently accurate that she will modify her angle of dance on the “dance floor” of the hive to compensate for the apparent movement of the sun across the sky.

Bees’ sense of time is also extraordinarily accurate. Bees know precisely where the sun should be in the sky at any time of the day or night. This is important for our studies as this information can be determined simply by observing their daily behaviours.

These behaviours can thus provide an excellent indication of time perception. By measuring the direction a bee flies with respect to the sun, we can infer the time of day that the bee thinks it is.

In addition, adult bees display strong circadian rhythms in their daily flight activity. These are driven by a set of clock genes whose DNA sequences and expression patterns are closely related to mammals. Oscillations in the expression of clock genes drive daily rhythms in physiology and behaviour.

We have taken advantage of bees’ sense of time to determine the effects of general anaesthesia on time perception. Using state-of-the-art harmonic radar tracking technology, we investigated the effect of the anaesthetic isoflurane on bee sense of time using time-compensated sun-compass orientation as a marker.

Harmonic radar tracking makes it possible to track bees as they fly with unparalleled resolution. The system works by sending a signal that excites a miniaturised transponder attached to the thorax of the bee (Fig. 2). When excited by the radar signal, the transponder emits the first harmonic to which the receiver on the radar is specifically attuned. Reflected signals from other obstacles, such as the ground and trees, is filtered out. This system can accurately determine the position of the bees and therefore the path of their flights over a 2 km radius.Figure 2. The harmonic radar system used to track bee flights.
(a) Depiction of the radar system showing the two radar dishes (lower transmitting and upper detection dishes) and signals sent to and received from a transponder attached to a bee’s thorax (b).

With this system we tested the effects of extended anaesthesia on the bees’ sense of time. We trained bees to an artificial flower 400 meters east of their hive in a large open grass field in Brandenburg, Germany. Once trained under a cloudless sky, bees were captured while departing the artificial flower and were treated with isoflurane for 6 hours before being let go at a release site 500 metres to the south of the flower (Fig. 3).

Figure 3. Bees trained to fly from the hive to an artificial flower placed 400 metres to the east were captured at the flower, anaesthetised and released. If anaesthesia had no effect on their time perception, the bees should fly in the direction of the hive. If, however, their time perception was halted during anaesthesia the bees would be expected to make an error of 90° (i.e. fly north) after 6 hours of anaesthesia (“predicted shift” arrow). The average flight observed was 60° (“observed shift” arrow).

Before starting these experiments we made some predictions. If their sense of time was unaffected by anaesthesia, the anaesthetised bees would fly in a direction that would return them directly to the hive. But if their sense of time was halted, we predicted that their flight direction would be changed.

In the Northern Hemisphere this shift should occur in a clockwise direction, whereas in the Southern Hemisphere it should occur in an anticlockwise direction because the sun moves through the southern part of the sky in the Northern Hemisphere whereas it moves through the north in the Southern Hemisphere.

Given that the sun moves 15° per hour across the sky on average, we could also predict the size of the anticipated shift. Following a 6-hour anaesthetic, we calculated that if time perception was halted altogether, bees would show a 90° shift in their orientation angle (“predicted shift” arrow, Fig. 3).

We were excited by the results of our radar experiments, as they showed that bees anaesthetised for 6 hours do show a shift in sun-compass orientation, and that the shift was in the predicted clockwise direction for the Northern Hemisphere. The average size of the shift was somewhat less than predicted at 60° (“observed shift” arrow, Fig. 3), but we were sufficiently encouraged to conduct similar experiments in the Southern Hemisphere (where a counter clockwise shift was predicted).

The results from our work in New Zealand added further support to our Northern Hemisphere data, with an anaesthesia-induced anticlockwise shift in sun-compass orientation of a similar magnitude to the Northern Hemisphere results.

Convinced that these experiments were showing a robust and quantifiable effect of anaesthesia on time perception, we then turned our attention to the question of why time perception is altered. This was done at both a behavioural and molecular level.

After working out how to induce anaesthesia in a whole hive of approximately 10,000 bees, we conducted experiments with bee colonies maintained in the laboratory in time isolation. Time isolation was achieved by keeping the hive in an environmentally controlled chamber maintained at a constant temperature and with constant dim light. The hive was connected to a flight chamber containing an artificial flower, a water source and a pollen source, and we were able to measure the activity of bees as they entered and left the flight chamber.

We found that bees kept in time isolation continued to show strong circadian rhythms of locomotor activity (Fig. 4a), and this behavioural rhythm was used as our marker of an output from the honey bee’s circadian clock.

Our prediction in these experiments was that if anaesthesia affected time perception by acting on the circadian clock, we should see a shift in the phase of behavioural activity rhythms that match the shift in time perception.

Following a 6-hour anaesthetic administered at the same time of the day as the time perception studies, the bees continued to show strong rhythms in flight activity (Fig. 4a). There was, however, a large shift in the phase of these rhythms to a later time zone (Fig. 4a, days 6–9).

Strikingly, the size of the shift in circadian rhythms in the laboratory was very similar to the shift in orientation angle in the field. Following anaesthesia the bees shifted to a later time zone by approximately 4.3 hours, which equates to a shift of 64.5° in orientation angle.

By subsequently examining the levels of expression of two clock genes in bees (cryptochrome and period) we also showed an anaesthesia-induced phase delay of the molecular clock (Fig. 4b). A 6-hour anaesthetic caused a delay in the expression of these genes by 4.9 hours (cryptochrome) and 4.3 hours (period).Figure 4. The effect of anaesthesia on behavioural and clock gene rhythms. (a) Hive activity after a 6-hour general anaesthetic (purple block) shifts 4.3 hours later in the day (as indicated by the differeence between the blue dashed line and the red line). (b) The effect of the same anaesthetic on the expression of two clock genes: period and cryptochrome. The blue line is the rhythm before the anaesthetic and the red line shows these rhythms shifting after anaesthesia.

The reason for the behavioural shift can thus be directly attributed to the anaesthetic acting on the clock at a fundamental biochemical level. The data from our different experiments were in strong agreement.

These results provide, for the first time, a definitive and quantitative explanation of how and why time perception is altered following anaesthesia. The strange feeling that time has not passed after an anaesthetic is because isoflurane acts on the central circadian clock in the brain, shifting it to a later time zone and effectively causing chemically-induced jet-lag. Furthermore, the extent of the circadian clock shift appears to directly determine the extent of the subsequent change in time perception.

We then questioned whether this anaesthesia-induced jet-lag occurs at different times of the day and the night. Intriguingly it does not. When bees were anaesthetised during the night there was no effect on the clock, either at a behavioural or at a molecular level. This is common to agents that affect the circadian clock.

These results, of course, led us to further questions. How long does the effect persist in the presence of strong daily time cues? Can it be prevented? Does this happen in humans? These are all questions we are working on.

Further bee studies in which we have tracked the timing of daily nectar feeding using radiofrequency identification system tags (similar to security cards used for accessing buildings) shows that visits to artificial flowers are delayed for at least 3 days after a 6-hour general anaesthetic despite the exposure of bees to strong daily light cycles. However, the magnitude of the shift in light cycles is much less than the 3–4-hour shift seen in time isolation. This finding has led us to investigate whether we might be able to reduce the size of anaesthesia-induced jet-lag by administering light and anaesthesia together.

An important extension of our findings is how it might impact on humans. Given the similarity of the circadian clocks of honey bees and mammals it may be reasonable to assume that anaesthesia-induced jet-lag is one of the underlying causes of post-operative sleep disruption in humans. Addressing the effects of general anaesthesia on the circadian clock may thus provide a way to speed up postoperative recovery.

Guy Warman is a senior lecturer and James Cheeseman a lecturer in the Department of Anaesthesiology at the University of Auckland. Craig Millar is a senior lecturer in the School of Biological Sciences at the University of Auckland. Eva Winnebeck, Randolf Menzel and Jamie Sleigh were major contributors and authors of this research, which was funded by a Marsden Grant from the Royal Society of New Zealand and by the University of Auckland, and published in Proceeedings of the National Academy of Sciences.